The primary goal of this Phase I STTR proposal is to develop a prototype of a rapid molecular diagnostics test to improve prediction of the antibiotic resistance of uropathogenic. Escherichia coli by determining their clonal identity (clonotype). The clonotype determination will be done by detection of the presence/absence of a limited number (d10) of single nucleotide polymorphisms (SNPs) that will be selected from 8 genetic loci used for the clonal typing of E. coli - seven loci from the standard multi-locus sequence typing (MLST) scheme for E. coli and fast-evolving gene for the type 1 fimbrial adhesin, fimH. The SNPs binary interrogation could be performed in either thermocycler or isothermal DNA amplification protocols using a simple single-plex configuration adapted to the standard 8-/12-tube PCR strips and common instrumentation platforms. These tests will be designed to perform in near-patient settings such as clinical laboratories in emergency rooms, urgent care clinics and/or hospitals. The test will be done using patients' urine in a cost-effective and timely manner (<30 min), with the goal to improve the empirical (pre-antibiogram) choice of antibiotic treatment, reducing potential 'drug-bug' mismatch and overuse of last-line broad-spectrum antimicrobials. To achieve the goals, we will determine MLST/fimH and antibiogram profiles of a comprehensive number of clinical isolates and establish the association between resistance to commonly used antibiotics and the clonal identity of pathogens. Phylogenetically-unlinked (homoplasic) SNPs that are distributed across the clonotypes in a relatively random and even fashion will be identified. Alternate combinations of 6 to 10 SNPs will be analyzed for the ability to discriminate up to 100 of the most critical (size- and resistance-wise) clonotypes of E. coli. Oligonucleotide primers capable of robust interrogation of the SNP combinations will be designed and validated for different amplification protocols. The proposed studies will be performed as collaboration between ID Genomics, Inc, the University of Washington's start-up company specializing in sequence-based typing of microbial pathogens, and the University of Washington, Seattle, laboratory of Evgeni Sokurenko (the lead PI).